Technical Abstract:
Background: Multi drug resistant (MDR) Salmonella enterica is found in food animals and may consequently pose a risk to humans through food borne transmission. To understand the mechanisms that drive this problem, the genetic elements associated with MDR need to be determined. These MDR elements in Salmonella are often associated with transmissible plasmids.
Methods: MDR Salmonella isolated from healthy animals at slaughter were screened by multiplex Polymerase Chain Reaction for 18 plasmid replicon types. The isolates were further screened by DNA microarray analysis for 1,269 resistance and plasmid genes. From these results, 12 isolates with the most diverse plasmid replicons and antimicrobial resistance genes detected were selected for plasmid DNA analysis by 454 pyro-sequencing.
Results: Plasmid sequencing identified genes, plasmids, and genetic elements consistent with the polymerase chain reaction (PCR) and microarray data. These included multiple alleles of resistance genes such as blaCMY-2, blaTEM-1, floR, aac(3), aadA, aphA1, strA, strB, sulI, sulII, dfrA, dhf, tet(A)(B)(C)(D), and tetR. Plasmid backbone genes detected were homologous to replication, maintenance, and conjugative transfer genes found in several plasmid replicon groups. Long contiguous DNA sequences were assembled from the data by further analysis. Sections of these sequences (up to 30,000 bp long) were nearly 100% homologous to antimicrobial resistance and virulence plasmids found in the National Center for Biology Information database. These included members of the IncA/C, I1, HI1, F, and P plasmid replicon types. Other contiguous sections had less homology, or homology to sequences found on other genetic elements, or limited homology to any known sequences indicating high levels of variability in these Salmonella MDR plasmids.
Conclusions: These data demonstrate that plasmids carrying resistance genes in Salmonella are highly diverse in their replicon types, gene content, and overall structure. In addition to having high variability in the antimicrobial resistance gene cassettes, plasmid backbone regions are also variable and some plasmids may be chimeras of multiple plasmid backbones. Sequence analysis of these MDR plasmids improves our knowledge of the genetic mechanisms responsible for the spread of MDR Salmonella.